The Immunosuppressant FK506 Uncovers a Positive Regulatory Cross-talk between the Hog1p and Gcn2p Pathways

Abstract

The immunosuppressant Tacrolimus (FK506) has increased the survival rates of organ transplantation. FK506 exerts its immunosuppressive effect by inhibition of the protein phosphatase calcineurin in activated T-cells. Unfortunately, FK506 therapy is associated with undesired non-therapeutic effects involving targets other than calcineurin. To identify these targets we have addressed FK506 cellular toxicity in budding yeast. We show that FK506 increased cell sensitivity upon osmotic challenge independently of calcineurin and the FK506-binding proteins Fpr1p, -2p, -3p, and -4p. FK506 also induced strong amino acid starvation and activation of the general control (GCN) pathway. Tryptophan prototrophy or excess tryptophan overcame FK506 toxicity, showing that tryptophan deprivation mediated this effect. Mutation of the GCN3 and -4 genes partially alleviated FK506 toxicity, suggesting that activation of the GCN pathway by FK506 was also involved in osmotic tolerance. FK506 enhanced osmotic stress-dependent Hog1p kinase phosphorylation that was not accompanied by induction of a Hog1p-dependent reporter. Interestingly, deletion of the GCN2 gene suppressed FK506-dependent Hog1p hyperphosphorylation and restored Hog1p-dependent reporter activity. Conversely, deletion of the HOG1 gene impaired FK506-dependent activation of Gcn2p kinase and translation of a GCN4-LacZ reporter, highlighting functional cross-talk between the Gcn2p and Hog1p protein kinases. Taken together, these data demonstrate that both FK506-induced amino acid starvation and activation of the GCN pathway contribute to cell sensitivity to osmotic stress and reveal a positive regulatory loop between the Hog1p and Gcn2p pathways. Given the conserved nature of Gcn2p and Hog1p pathways, this mechanism of FK506 toxicity could be relevant to the non-therapeutic effects of FK506 therapy.

Miconazole Induces Changes in Actin Cytoskeleton prior to Reactive Oxygen Species Induction in Yeast

“…Survival of the yeast culture after 5 h of incubation with 5 g/ml miconazole was 44%, whereas addition of 0.1-5 mM Trp increased the yeast cell survival to up to 100%. These data corroborate earlier findings regarding alleviation of the growth inhibitory effect of an antifungal compound by Trp (10,11). In these studies, either 0.4 or 5 mM Trp was used.…”

ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum

“…Toxicity could not be relieved by an excess of tryptophan (data not shown). Therefore, this may be a different phenomenon from that described in yeasts and mammals using FK506 (Rodriguez-Hernandez et al, 2003;Sanchez-Perez et al, 2004). This result raises the possibility of the HOG and Ca 2+ pathways acting over common processes (Maeta et al, 2005), since only overexpression of hog1 F315S can restore, at least in .…”

“…In yeast, the calcineurin inhibitor FK506 increases osmotic sensitivity by inducing amino acid starvation (Rodriguez-Hernandez et al, 2003). However, when using CsA in T. harzianum, no changes in stress resistance were observed (Fig.…”

Mecanismos moleculares involucrados en la citotoxicidad del agente antitumoral beta-lapachona.

“…Estos datos sugieren que: i) Gcn2p parece activarse por situaciones de estrés que no llevan a ayuno de nutrientes, y ii) la función de Gcn2p en respuesta a la droga no es regular la traducción de proteínas. Estos datos están en acuerdo con evidencias previas que asignan a Gcn2p una función no traduccional (Rodríguez-Hernández et al, 2003).…”

“…Sin embargo, no puede descartarse que algún otro componente de la maquinaria de control/reparación implicado en la respuesta a tratamiento con betalapachona esté sujeto a control traduccional, o algún otro regulador de ciclo no conectado directamente con el control de daño al ADN. A este respecto, se ha reportado previamente una conexión entre Gcn2p y Hog1p (Rodríguez-Hernández et al, 2003) que sustenta esta posibilidad.…”